Analysis of In Vivo Transcription Factor Recruitment by Chromatin Immunoprecipitation of Mouse Embryonic Kidney

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful technique for examining transcription factor recruitment to chromatin, or histone modifications, at the level of specific genomic sequences. As such, it provides an invaluable tool for elucidating gene regulation at the molecular level. Combined with high-throughput methods such as second generation sequencing (ChIP-Seq), this technique is now commonly used for studying DNA–protein interactions at a genome-wide scale. The ChIP technique is based on covalent cross-linking of DNA and proteins with formaldehyde, followed by chromatin fragmentation, either enzymatic or by sonication, and immunoprecipitation of protein–DNA complexes using antibodies specific for the protein of interest. The immunoprecipitated DNA is then purified and the DNA sequences associated with the immunoprecipitated protein are identified by PCR (ChIP-PCR) or, alternatively, by direct sequencing (ChIP-Seq). Initially, the vast majority of ChIP experiments were performed on cultured cell lines. More recently, this technique has been adapted to a variety of tissues in different model organisms. We describe here a ChIP protocol on freshly isolated mouse embryonic kidneys for in vivo analysis of transcription factor recruitment on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.